Molecules suitable for binding to a metal layer for covalently immobilizing biomolecules

ABSTRACT

An article is provided for immobilizing functional organic biomolecules (e.g. proteins, DNA, and the like) through a covalent bond to a thiolate or disulfide monolayer to a metal surface wherein an extra activation step of the surface layer or an activation step of the functional biomolecules or bioreceptors could be avoided. The monolayer can contain, but is not limited to, two moieties. One has a group that resists nonspecific adsorption and another has a group that directly (without activation) reacts with functional groups on the biomolecules. In addition, poly(ethylene oxide) groups are incorporated in the monolayer surfaces to resist the nonspecific adsorption and to enhance the specific affinity interactions. A sensor device including these monolayers is also provided to perform reproducible, sensitive, specific and stable bioanalysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/637,390filed Dec. 12, 2006, which claims the benefit under 35 U.S.C. §119(e) ofU.S. provisional application Ser. No. 60/751,383, filed Dec. 16, 2005,the disclosures of which are hereby expressly incorporated by referencein their entirety and are hereby expressly made a portion of thisapplication.

FIELD OF THE INVENTION

The preferred embodiments relate to the field of sensors, particularlybiosensors for detecting an analyte in a sample, especially sensorscomprising a metal layer substrate and one or more organic moleculesbound to the substrate. The preferred embodiments relate as well to themanufacture of such sensors, and organic molecules suitable for use insuch sensors.

BACKGROUND OF THE INVENTION

In the past two decades, the biological and medical fields havediscovered the great advantages in the use of biosensors and biochipscapable of characterizing and quantifying (bio)molecules. The fastestgrowing area in biosensors research involves affinity-based biosensorsor immunosensors. These sensors are expected to revolutionize in areaslike diagnostics, food processing, antiterrorism, environmentalmonitoring and public health where rapid detection combined to highsensitivity are important.

The development of strategies to immobilize groups of biomolecules tosubstrates has given rise to the field of biochips and has dramaticallyincreased the rate and scope of discoveries in basic and appliedscience. A key challenge in biochip technology has been the developmentof reliable and reproducible chemistries for the immobilization ofligands or bioreceptors to a single substrate.

In order to be broadly useful for the preparation of a wide variety ofbiochips, the immobilization reaction should have severalcharacteristics. First, the reaction should occur rapidly and thereforeallow the use of low concentrations of reagents for immobilization.Second, the chemistry should require little, if any, post-syntheticmodifications of ligands before immobilization to maximize the number ofcompounds that can be generated by solution or by solid-phase synthesisand minimize the cost of these reagents. Third, the immobilizationprocess should occur selectively in the presence of common functionalgroups, including amines, thiols, carboxylic acids, and alcohols, toensure that ligands are immobilized in a preferable oriented andhomogeneous manner. Finally, the reaction should have well-behavedkinetics and be easily monitored with conventional spectroscopic methodsto control the density of ligands on the chip.

Several chemical systems have been described and used for theimmobilization of proteins to solid biochip surfaces. The proteincoupling chemistry depends upon the underlying substrate of the biochipcombined with the desired bioreceptor species one would like to coupleto the biochip substrate. A number of methods have therefore beenapplied for the immobilization of receptor biomolecules, e.g.adsorption, covalent attachment to silanes or mixed layers of thiols,embedding in polymers and membranes. These different kinds ofchemistries should be a compromise between the functional groupsavailable on these chemistries and the functional groups available onthe bioreceptor species, which one wants to immobilize on the biochipsubstrate. The groups on the bioreceptor species are important toachieve a random or orientated immobilization of the immobilizedbioreceptors

It is known in the art of biosensors that molecules having the formulaX—R—Ch-M adhered to a surface as part of a self assembled monolayer,where X is a functionality that adheres to the surface, R is a spacermoiety, M is a metal and Ch is a chelating agent for the metal ion M.These monolayers only have a limited surface accessibility forbiological binding and oriented immobilization. Moreover, this type ofmonolayer can only be achieved via an extra cross-linking step.

A method for immobilizing proteins on mixed self-assembled monolayers ofalkanethiols is also known in the art. This method needs an activationstep comprising forming an N-hydroxysuccinimidyl (NHS) ester from thecarboxylic acid groups of the self-assembled monolayer and then couplingthis ester to a free amino group of the protein. In a first step, aself-assembling monolayer having free carboxylic acid groups is formedonto a gold surface. In a next step, the surface carboxylic acid groupsare activated to form the NHS ester, followed by displacement of the NHSester with an amino group of the protein to form an amido function.Since several steps have to be performed after deposition of theself-assembling monolayer onto the substrate, the yield reduces aftereach step, resulting in a low final yield of the immobilization degree.

Self-assembled monolayers with a CH═CH₂ end group are also known in theart but no biomolecules or bioreceptors have been coupled to thisfunctional group on a gold surface. In addition, such monolayers do notincorporate poly(ethylene oxide) groups which are desirable to avoidnon-specific adsorption, a key issue in biosensing experiments.

Thiolate or disulfide self-assembled monolayers with aldehyde or epoxyend groups, which can be directly coupled to functional groups onbiomolecules or bioreceptors are also known in the art. However theseself-assembled monolayers do not incorporate poly(ethylene oxide) groupsallowing a better sensitivity and specificity of the final biosensorinterface.

Self-assembled monolayers with pre-activated groups, e.g.dithio-bis(succinimidylundecanoate), dithio-bis(succinimidylpropionate)and dithio-bis(succinimidylhexadecanoate) are already known in the artand can incorporate poly(ethylene oxide) groups. However theN-hydroxysuccinimidyl group is very sensitive to hydrolysis, which makesit difficult to store these activated samples before use.

Self-assembled monolayers incorporating poly(ethylene oxide) groups anda preactivated maleimidyl group are also known in the art. However amaleimide reacts preferentially with free thiol groups, which are notalways readily available in proteins such as antibodies. In order toavoid this drawback, antibodies can be reduced to generate free thiolgroups but this additional step is difficult to perform when formingbiochips and often decreases the antibody affinity.

Health and environment related fields, faces various biochemicalprocesses, which have to be evaluated rapidly at decreasing detectionlevels. Many biochemical analytical methods involve immobilization of abiological molecule on a surface. The increasing miniaturization and thedemand for sensitivity require a covalent immobilization ofbiomolecules. Affinity biosensor transducers are defined as systemscontaining at least one biological element able to recognize an analyte.This element is called the biological recognition layer and consists ofa probe molecule, covalently bound to a linking layer, which makes theconnection with the transducer. The substrate can be a deposit of ametal film on any convenient support or any other solid surface able toselectively bind monolayers. Preferred metals include gold, silver,Ga—As alloys, palladium, platinum, copper, and the like. Silanes andalkyl phosphate monolayers can also be used on oxide material substrateslike SiO₂, Nb₂O₅, TiO₂, ZrO₂, Al₂O₃, and Ta₂O₅. A biosensor must respondto major qualities like stability, specificity, selectivity, andreproducibility. For all those reasons, only few affinity biosensors arecommercially available. The major challenge is the realization of newspecific and selective self-assembled monolayers and the receptors. Ananalyte must be detectable in an excess of other proteins. The mostcommon receptors are antibodies and specific binding proteins which havea reversible specific binding affinity for an analyte. Chemicalmodifications of the surface moieties may create new surfacefunctionalities, such as, for example, amine-terminated functionalgroups appropriate for particular diagnostic or therapeutic operations.

SUMMARY OF THE INVENTION

New organic molecules are provided suitable for forming aself-assembling monolayer onto a surface, in particular a metal surface,while solving one or more of the problems identified herein above. Amethod is also provided for forming mixed self-assembled monolayers ofthiols or disulfide molecules incorporating poly(ethylene oxide) groupsand two functional groups, preferably wherein one functional group ispre-activated and can be directly and covalently coupled to the aminogroups of a bioreceptor while the other functional group providesresistance to non-specific adsorption. Organic molecules are providedthat can form self-assembling monolayers suitable for making highselectivity, high stability and/or high reproducibility sensorsubstrates linked to a recognition molecule. In addition these organicmolecules should decrease non-specific binding and allow binding of areceptor molecule onto the self-assembling monolayer in one single step.

In a first aspect, an organic molecule is provided having the structuralformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂, wherein:

-   -   X₂ is either H or S—R₅,

R₅ is an organic spacer selected from the group consisting of

-   -   —R₂—(O—CH₂—[CH₂]_(t))_(n)—O—(CH₂)_(c)—X₁′ and        R₃—(O—CH₂—[CH₂]_(t))_(n)—Y₁,    -   t is 1 or 2,    -   c is an integer from 0 to 3,    -   n is an integer from 3 to 15,000,    -   R₁, R₂ and R₃ are each independently a saturated or        ethylenically unsaturated hydrocarbyl group with 3 to 30 carbon        atoms selected from the group consisting of alkyl, alkenyl,        cycloalkyl, cycloalkyl-alkyl, cycloalkenyl, cycloalkenylalkyl        and cycloalkylalkenyl, said group optionally comprising one or        more heteroatoms selected from nitrogen, oxygen and sulfur in        the main chain, and said group optionally comprising one or more        oxo substituents,    -   Y₁ is either hydroxy or methoxy,    -   X₁ and X₁′ are each independently selected from the group        consisting of fluorophenyl, fluorobenzoyl,        fluorophenoxycarbonyl, nitrophenoxycarbonyl, oxiranyl,        aziridinyl, C₂₋₁₂ alkenyl, imino-ether, dichlorotriazinyl,        sulfonyl halide, alkoxycarbonyl, isothiocyanato, isocyanato,        carbonyl halide, haloalkylcarbonyl, carboxylic acid anhydride,        diazonium carbonyl, N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

In one embodiment of the first aspect, X₁ represents a group which candirectly react with a biomolecule, i.e. without prior activation ofeither the organic molecule of preferred embodiments or the biomoleculeto be immobilized. In a preferred embodiment, the biomolecule has atleast one primary amino-group, which can react with the X₁ group. Inanother preferred embodiment, the amino group of a lysine amino acid ofa protein/peptide or the amino group at the end of a DNA or RNA strandor oligo nucleic acid chain can react with the moleculeX₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ at the X₁ side of themolecule.

The group —([CH₂]_(t)—CH₂—O)_(n)— inX₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ and inR₂—(O—CH₂—[CH₂]_(t))_(n)—O—(CH₂)_(c)—X₁′ is preferably selected in sucha way that non-specific adsorption of a biomolecules to the organicmolecule of preferred embodiments is substantially avoided.

X₁ and X₁′ each independently include a chemical group compatible withmonolayer formation and which needs no in situ activation prior toreaction with the biological moiety.

In a second aspect, a device for immobilizing at least one biomoleculethrough a covalent bond is provided, said device comprising:

-   -   a substrate comprising a metal layer,    -   one or more first species bound onto said metal layer, wherein        each of said one or more first species is a molecule having a        structural formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M,        wherein    -   M is said metal layer to which said one or more molecules are        bounded,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   c is an integer from 0 to 3    -   R₁ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   X₁ is selected from the group consisting of fluorophenyl,        fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,        oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,        dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,        isothiocyanato, isocyanato, carbonyl halide, haloalkylcarbonyl,        carboxylic acid anhydride, diazonium carbonyl,        N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

In an embodiment of the second aspect, the device further comprises asecond species, the second species being bound onto the metal layer andincluding a compound having the chemical formula:

Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M

wherein:

-   -   R₃ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   M is the metal of said metal layer, and    -   Y₁ is either a hydroxy group or a methoxy group.

The second species is preferably selected such that non-specificadsorption of biomolecules is substantially avoided.

In a third aspect, a process for preparing a device according to thesecond aspect is provided. This process comprises the steps of:

-   -   a) providing a metal layer,    -   b) contacting said metal layer with one or more molecules        according to the first aspect, wherein said one or more        molecules self-assemble to form a layer of one or more first        species bound onto said metal layer, wherein each of said one or        more first species is a molecule having a structural formula        X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M, wherein    -   M is said metal layer to which said one or more molecules are        bounded,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   c is an integer from 0 to 3,    -   R₁ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   X₁ is selected from the group consisting of fluorophenyl,        fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,        oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,        dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,        isothiocyanato, isocyanato, carbonyl halide, haloalkylcarbonyl,        carboxylic acid anhydride, diazonium carbonyl,        N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

In a fourth aspect, a process for preparing a device according to anembodiment of the second aspect is provided, comprising the steps of:

-   -   a) providing a metal layer,    -   b) contacting said metal layer with one or more molecules        according to the first aspect and with one or more molecules        having a formula Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂,    -   wherein said one or more molecules according to the first aspect        and said one or more molecules having a formula        Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂ self-assemble to form a mixed        layer of one or more first species and one or more second        species bound onto said metal layer, wherein each of said one or        more first species is a molecule having a structural formula        X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M and each of said one        or more second species is a molecule        Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M wherein:        -   Y₂ is either H or —S—R₄—(O—CH₂—[CH₂]_(t))_(n)—Y₁′,        -   R₁, R₃ and R₄ are each independently a saturated or            ethylenically unsaturated hydrocarbyl group with 3 to 30            carbon atoms selected from the group consisting of alkyl,            alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,            cycloalkenylalkyl and cycloalkylalkenyl, said group            optionally comprising one or more heteroatoms selected from            nitrogen, oxygen and sulfur in the main chain, and said            group optionally comprising one or more oxo substituents,        -   Y₁ and Y₁′ are independently either a hydroxy group or a            methoxy group,        -   M is said metal layer to which said one or more molecules            are bounded,        -   t is an integer from 1 to 2,        -   n is an integer from 3 to 15,000,        -   c is an integer from 0 to 3, and        -   X₁ is selected from the group consisting of fluorophenyl,            fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,            oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,            dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,            isothiocyanato, isocyanato, carbonyl halide,            haloalkylcarbonyl, carboxylic acid anhydride, diazonium            carbonyl, N-(2-oxotetrahydro-3-thienyl)amido and            N-carboxy-thiazolidinyl-2-thione.

In a fifth aspect, a sensor is provided for the detection of an analytein a sample fluid, the sensor comprising:

-   -   a device according to the second aspect on which at least one        biomolecule is immobilized by a covalent bound, and    -   a transducer.

In a sixth aspect, a method for manufacturing a sensor according to thefifth aspect is provided, wherein the method comprises the steps of:

-   -   contacting a device according to the second aspect with a        solution of at least one biomolecule,    -   connecting said device to a transducer.

In an embodiment of this sixth aspect, the at least one biomolecule hasat least one primary amino group. In another embodiment of this sixthaspect, the device and/or said at least one biomolecule is notchemically activated prior to contacting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents schematically a device according to an embodiment ofthe second aspect.

FIG. 2 shows cyclic voltammograms (CV) of a gold layer with and withoutmolecules according to an embodiment.

FIG. 3 shows a surface plasmon resonance (SPR) signal recorded by asensor according to an embodiment.

FIG. 4 shows SPR data comparing the efficiency of binding of ananti-body to a device according to a preferred embodiment and to adevice according to the prior art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

As used herein and unless stated otherwise, the term “self-assembling”relates the association of molecules without guidance or management froman outside source. It results in a layer, usually a monolayer of thosemolecules on a surface.

As used herein and unless stated otherwise, the terms “metal layer”relates to a layer made from one or more metals or metal alloys, havinga thickness of from 1 nm to 10 nm (but not limited hereto) and which canbe either self supported or supported by a substrate. If the layer isself-supported, it can be named “substrate” as well.

As used herein and unless stated otherwise, the term “hydrocarbyl group”relates to a saturated or ethylenically unsaturated, cyclic ornon-cyclic chain selected from the group consisting of alkyl, alkenyl,cycloalkyl, cycloalkenyl cycloalkyl-alkyl, cycloalkenyl-alkyl andcycloalkyl-alkenyl; when non-cyclic, this chain can be linear orbranched.

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those skilled in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

In a first aspect, an organic molecule is provided with the structuralformula:

X₁-PEO1-R₁—S—X₂

wherein:

-   -   X₂ is hydrogen, —S—R₂—PEO2-X₁ or —S—R₂-PEO2-X₁′, and    -   X₁ and X₁′ are as defined herein-above, and    -   R₁ and R₂ are selected such that a stable ordered monolayer is        formed.        R₁ and R₂ independently include a spacer. R₁ and R₂ can be the        same or different. R₁ and R₂ can have the same chemical        composition such that a symmetrical molecule is formed.        Symmetrical molecules have generally the advantage of a more        straightforward synthesis. R1 and R2 preferably promote the        formation of a self-assembling monolayer and can be a        hydrocarbon chain, i.e. a hydrocarbyl group. The hydrocarbyl        group can include n carbon atoms, n being an integer higher (or        equal to) 3, 6, 8, or 10, preferably from 3 to 30. The spacer        can also represent a hydrocarbyl group interrupted by a        —CO-(ketone), —CONH, —CONHCO—, —CSNH—, —CS—, and the like. For        instance the hydrocarbyl group can optionally comprise one or        more carboxy groups in the main chain of the hydrocarbyl group.        The hydrocarbyl group can also be interrupted by one or more        heteroatoms. The heteroatom can be selected from the group        consisting of —N—, —O—, and —S—. In particular, the heteroatom        can be O. For instance, R₁ and R₂ can independently comprise one        or more heteroatoms in the main chain of the hydrocarbyl group.        The hydrocarbyl group can also be branched. The spacer can        include a first part which is a hydrocarbon chain and a second        part which is a hydrocarbyl group interrupted by a heteroatom        such as oxygen.

In an advantageous embodiment, R₁ and R₂ independently include an alkylchain —(CH₂)_(n), n being an integer from 3 to 30, for instance from 3to 25, preferably from 3 to 20, e.g. from 5 to 20, or from 8 to 16. Inanother advantageous embodiment, n is an integer. In a preferredembodiment n is higher than 4, higher than 6, higher than 8, higher than10, higher than 11, higher than 12, higher than 13, higher than 15 orhigher than 20. Alternatively, n is an integer from 8 to 16, from 10 to16.

In a preferred embodiment R₁ and R₂ are each independently a saturatedor ethylenically unsaturated hydrocarbyl group with 3 to 30 carbon atomsselected from the group consisting of alkyl, alkenyl, cycloalkyl,cycloalkyl-alkyl, cycloalkenyl, cycloalkenylalkyl and cycloalkylalkenyl,said group optionally comprising one or more heteroatoms selected fromnitrogen, oxygen and sulfur in the main chain, and said group optionallycomprising one or more oxo substituents.

PEO1 and PEO2 independently include a —(O—CH₂—[CH₂]₁)_(n)—O— group, nbeing an integer from 3 to 15,000, preferably from 3 to 1250. PEO1 andPEO2 are preferably selected such that non-specific adsorption ofchemical molecules is avoided. PEO1 and PEO2 independently include(O—CH₂—[CH₂]_(t))_(n)—O— such as e.g. (O—CH₂—CH₂—)_(n)—O—, n being aninteger from 3 to 15,000, preferably from 3 to 1250, from 3 to 1000,from 3 to 500, from 3 to 100, from 3 to 50, from 3 to 30, from 3 to 20,from 3 to 15, from 3 to 10, from 3 to 8, or from 2 to 8. Alternatively,n is an integer from 1 to 30, from 1 to 20, from 1 to 15, from 1 to 10,from 1 to 8, or from 1 to 6. t is an integer of from 1 to 10, preferably1 or 2.

In a particular embodiment, PEO1 is represented by the structuralformula —(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n) and PEO2 is independentlyfrom PEO1 represented by the structural formula—(O—CH₂—[CH₂]_(t))_(n)—O—(CH₂)_(n)—, n being an integer and c being aninteger. The groups of formula —(O—CH₂—[CH₂]_(t))_(n) are intended foravoiding non-specific adsorption. The variable “n” is preferably from 1to 10, or more preferably from 1 to 8. The variable “c” is an integer offrom 0 to 3, preferably from 1 to 3. Preferably c can be 1, 2, or 3. Thevariable “t” is 1 or 2.

X₁ is a chemical group suitable for binding biological moieties (e.g. abiomolecule) onto a monolayer. More specifically, X₁ is a highlyreactive functional moiety compatible with monolayer formation and whichneeds no in situ activation prior to reaction with the biologicalmoiety. The biological moieties that are covalently bound or adsorbedonto the monolayer can be, but are not limited to, nucleic acid strands(DNA, PNA, RNA), proteins, hormones, antibiotics, antibodies, chemicallyor enzymatically modified antibodies, VHH fragments of lama antibodies,synthetic receptors, single chain Fv's, antigens, enzymes, drugs, drugsof abuse. In general, these biological moieties will serve as abiological sensing element and will be part of a sensor. The resultingsensor will be suitable for determining the presence of a compound, suchas a target molecule, which interacts with the biological sensingelement. The target molecule could be, but is not limited to,complementary nucleic acid strands (DNA, PNA, RNA), proteins, hormones,antibiotics, antibodies, antigens, enzymes, drugs, drugs of abuse ormolecules such as specific molecules present in for example gases andliquids.

X₁ includes preferably a chemical group selected from the groupconsisting of fluorophenyl, fluorobenzoyl, fluorophenoxycarbonyl,nitrophenoxycarbonyl, oxirane, aziridine, C₂₋₁₂ alkenyl, imino-ether,dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl, isothiocyanato,isocyanato, carbonyl halide, haloalkylcarbonyl, carboxylic acidanhydride, diazonium carbonyl, N-(2-oxotetrahydro-3-thienyl)amido andN-carboxy-thiazolidine-2-thione.

For instance, X₁ may include one of the following species:

wherein R is either CH₃, CH₂ or OCH₂CH₃.

X₁ is a chemical group for binding biological moieties to the moleculeas described in the first aspect. The chemical group X₁ is used forimmobilization of a molecule, e.g. a recognition molecule. Therecognition molecule can be e.g. a biomolecule. The recognitionmolecules can chemically interact (e.g. bind) to the group X₁ and inparticular the NH₂ group of a recognition molecule can bind to the groupX₁ or can react with the group X₁ to bind to moleculeX₁—(CH₂)_(n)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂.

More specifically, X₁ is a highly reactive functional moiety compatiblewith monolayer formation and which needs no in situ activation prior toreaction with the biological moiety. The biological moieties that cancovalently be bound or adsorbed to the chemical molecule as in the firstaspect can be, but are not limited to, nucleic acid strands (DNA, PNA,RNA), proteins, hormones, antibiotics, antibodies, chemically orenzymatically modified antibodies, VHH fragments of lama antibodies,synthetic receptors, single chain Fv's, antigens, enzymes, drugs, drugsof abuse. In general, these biological moieties will serve as abiological recognition molecule.

It should be understood that the scope of the molecule as described inthe first aspect comprises all possible combinations of chemical groupsand elements as specified herein.

In one particular embodiment, the molecule having the structural formulaX1-PEO1-R1-S—X2 corresponds to the structural formulaX1-(CH2)c-PEO1-R1-S—X2. In another embodiment, the structural formulaX1-PEO1-R1-S—X2 corresponds to the structural formulaX1-(CH2)c-O—([CH2]t-CH2-O)n-R1-S—X2 wherein

-   -   —X₂ is either H or S—R₅,    -   R₅ is an organic spacer selected from the group consisting of        R₂—(O—CH₂—[CH₂]_(t))_(n)—O—(CH₂)_(c)—X₁′ and        R₃—(O—CH₂—[CH₂]_(t))_(n)—Y₁,    -   t is 1 or 2,    -   c is an integer from 0 to 3,    -   n is an integer from 3 to 15,000,    -   R₁, R₂ and R₃ are each independently a saturated or        ethylenically unsaturated hydrocarbyl group with 3 to 30 carbon        atoms selected from the group consisting of alkyl, alkenyl,        cycloalkyl, cycloalkyl-alkyl, cycloalkenyl, cycloalkenylalkyl        and cycloalkylalkenyl, said group optionally comprising one or        more heteroatoms selected from nitrogen, oxygen and sulfur in        the main chain, and said group optionally comprising one or more        oxo substituents,    -   Y₁ is either hydroxy or methoxy,    -   X₁ and X₁′ are each independently selected from the group        consisting of fluorophenyl, fluorobenzoyl,        fluorophenoxycarbonyl, nitrophenoxycarbonyl, oxiranyl,        aziridinyl, C₂₋₁₂ alkenyl, imino-ether, dichlorotriazinyl,        sulfonyl halide, alkoxycarbonyl, isothiocyanato, isocyanato,        carbonyl halide, haloalkylcarbonyl, carboxylic acid anhydride,        diazonium carbonyl, N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

The functional group —S—S— or H—S— is able to adhere (i.e. chemisorb) toa surface such as a metal layer and can chemically interact (e.g. bind)with said metal layer. For instance, a covalent bond S-M, with S being asulfur atom and M being the metal, can be formed. The interactionbetween the sulfur atom and the substrate is well known to peopleskilled in the art.

In a second aspect, a device, suitable for the fabrication of a sensorin disclosed. The device comprises:

-   -   a substrate comprising a metal layer,    -   a first species being attached to the metal layer, the first        species including a chemical molecule as in the first aspect,        wherein the sulfur atoms of the chemical molecule bind to the        metal surface.

In an embodiment of this second aspect, a device for immobilizing atleast one biomolecule through a covalent bond is provided, the devicecomprising:

-   -   a substrate comprising a metal layer,    -   one or more first species bound onto said metal layer, wherein        each of said one or more first species is a molecule having a        structural formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M,        wherein    -   M is said metal layer to which said one or more molecules are        bounded,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   c is an integer from 0 to 3,    -   R₁ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   X₁ is selected from the group consisting of fluorophenyl,        fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,        oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,        dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,        isothiocyanato, isocyanato, carbonyl halide, haloalkylcarbonyl,        carboxylic acid anhydride, diazonium carbonyl,        N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

In another embodiment of the second aspect, the device as in the secondaspect further comprises a second species, said second species beingbound to the metal surface (i.e. the metal layer), the second speciesbeing obtained by contacting the metal layer with a compound of thestructural formula:

Y₁—R₃—S—Y₂

wherein:

-   -   Y₂ is hydrogen or —S—R₄—Y₁,    -   said sulfur atoms covalently bind to the metal layer, and    -   wherein R₃ and R₄ independently include a spacer including m        carbon atoms, m being an integer from 3 to 30,

The spacer promotes the formation of a self-assembling monolayer and maybe a hydrocarbon chain as defined herein above. R₃ and R₄ can have thesame chemical composition such that a symmetrical molecule is formed. Inan embodiment, R₃ and R₄ are each independently a spacer of m carbonatoms, m being an integer from 3 to 30, from 3 to 25, from 3 to 20, from5 to 20, or from 8 to 16. The total number of carbon atoms m ispreferably higher than 3, higher than 6, higher than 8, or higher than10.

Optionally, R₃ and R₄ may include q heteroatoms wherein (m+q) is aninteger preferably higher than 6. In a preferred embodiment, R₃ and R₄are independently selected from the group consisting of alkyl chains(CH₂)_(m) with m being an integer higher than 6 and alkyl chainsincluding q heteroatoms, q being an integer such that (m+q) is higherthan 6.

In another embodiment, R₃ and R₄ are each independently from each othera spacer including two parts, a first part for obtaining a stableordered monolayer and a second part for avoiding non-specificadsorption. In a particular embodiment, R₃ and R₄ are independently fromeach other (CH₂)_(e)—(CH₂—CH₂—O)_(f)—(CH₂)_(g), e being an integer, fbeing an integer and g being an integer. The alkyl chain is intended toachieve a stable ordered and reproducible system while the polyethyleneoxide groups are intended for avoiding non-specific adsorption. Thevariable “e” is preferably an integer from 1 to 20, from 5 to 20, from 5to 15, or from 5 to 12, e.g. 6 or 11. The variable “f” is preferably aninteger from 1 to 10, from 1 to 8, or from 2 to 6, e.g. 3, 4 or 5. Thevariable “g” is an integer preferably from 0 to 3, e.g. 1 or 2.

In other words, in this embodiment of the second aspect, the device asin the second aspect further comprising one or more second species boundonto said metal layer, wherein each of said one or more second speciesis a molecule having a structural formulaY₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M wherein:

-   -   R₃ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   M is the metal of said metal layer, and    -   Y₁ is either a hydroxy group or a methoxy group.

The first species and the second species are selected such that a mixedself-assembled layer such as a mixed self-assembled monolayer is formedon the metal layer. A mixed self-assembled monolayer results in bettersensitivity of the recognition molecule towards the target molecule inthe medium.

The molar ratio of the second species to the first species can be1000:1, 500:1, 100:1, 80:1, 70:1, 60:1, 50:1, 20:1, 10:1, 5:1, 95:5,90:10, 80:20, 70:30, or 60:40, and can be determined by spectroscopictechniques available to a person skilled in the art.

Non-specific adsorption is preferably avoided when the device is used asa sensor. Non-specific adsorption herein refers to interaction betweenthe recognition molecule immobilized at the surface and any speciesbeing present in a medium that preferably contains the target molecule.Such “any species” excludes the target molecule.

In advantageous embodiments, the first species has the structuralformula as in any of the examples. The substrate may comprise a metallayer comprising, but not limited hereto, gold, silver, mercury,aluminum, platinum, palladium, copper, cadmium, lead, iron, chromium,manganese, tungsten and alloys thereof. The substrate can be the metallayer itself. The substrate can also be a sensor, a biosensor, a DNAchip, a protein chip, a microarray, a microscope slide, a silicon waferor a microelectronic surface. The substrate can be a part of atransducer, which can be, but is not limited to, a Surface PlasmonResonance sensor, a Surface Acoustic Wave sensor, a Quartz CrystalMicrobalance, an amperometric sensor, a capacitive sensor, anInterdigitated Electrode or a ChemFET sensor. The surface can also havemagnetic properties such as a magnetic particle comprising a magneticmaterial such Fe₂O₃ or Fe₃O₄, optionally coated with a coating layer,preferably a metal coating layer.

The first species forms a self-assembling monolayer onto the surface ofthe device. Self-assembled monolayers are considered as a relativeordered assembly of molecules that spontaneously attach (or chemisorb)onto a surface. Molecules are preferably oriented parallel or preferablyunder an angle with respect to the surface.

Each group being part of a self-assembling monolayer preferably includesa functional group for attaching to the surface and a functional groupthat binds to the recognition molecule. In the preferred embodiments,the functional group being able to attach to the surface is a disulfidegroup —S—S— or a thiol group, and the functional group being able tobind a recognition molecule (e.g. to react with a recognition moleculein order to bind to the molecule) is the group X₁ as defined hereinabove. The functional group —S—S— or HS— is able to adhere (chemisorb)to a surface such as a metal and can chemically interact with the metalsurface. Interaction between the sulfur atom and the substrate is wellknown to people skilled in the art. The chemical group X₁ is used forsurface immobilization of a recognition molecule. The recognitionmolecules can be bound to this group and in particular, a NH₂ group(i.e. at least one primary amino group) of a recognition molecule canreact with the first species, i.e. with the group X₁ present on thisfirst species.

FIG. 1 represents schematically a device according to an embodiment ofthe second aspect. This device comprises a metal layer (2) on which afirst species (1) and a second species (3) are bound. The first and thesecond species form a monolayer (4) on the metal layer (2).

In a third aspect, a process for preparing a device according to thesecond aspect is provided. This process comprises the steps of:

-   -   a) providing a metal layer,    -   b) contacting said metal layer with one or more molecules        according to the first aspect, wherein said one or more        molecules self-assemble to form a layer of one or more first        species bound onto said metal layer, wherein each of said one or        more first species is a molecule having a structural formula        X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M, wherein    -   M is said metal layer to which said one or more molecules are        bounded,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   c is an integer from 0 to 3,    -   R₁ is a saturated or ethylenically unsaturated hydrocarbyl group        with 3 to 30 carbon atoms selected from the group consisting of        alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl,        cycloalkenylalkyl and cycloalkylalkenyl, said group optionally        comprising one or more heteroatoms selected from nitrogen,        oxygen and sulfur in the main chain, and said group optionally        comprising one or more oxo substituents,    -   X₁ is selected from the group consisting of fluorophenyl,        fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,        oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,        dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,        isothiocyanato, isocyanato, carbonyl halide, haloalkylcarbonyl,        carboxylic acid anhydride, diazonium carbonyl,        N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

Preferably, a treatment to remove any metal oxide layer present at thesurface of the metal layer is performed according to any method wellknown by the person skilled in the art.

FIG. 2 shows a cyclic voltammogram (CV) of a gold layer without amono-layer of molecules on its surface (Blanc, light line), a CV of thesame gold layer on which a mono-layer of molecules according to Example7 has been self-assembled (100% COpfp doted line) and a CV of the samegold layer on which a mono-layer of molecules according to Example 8 hasbeen self-assembled (100% COOpfp, dark line). The signal of the currentin CVs of the gold layers on which a mono-layer of molecules accordingto the preferred embodiments has been formed, show a dramatic decreasewhen compared with the signal of the current recorded for the goldsurface alone. This indicates a substantially complete coverage of thegold layer by the respective self-assembled mono-layers.

In a fourth aspect, a process is provided for preparing a deviceaccording to an embodiment of the second aspect, comprising the stepsof:

-   -   a) providing a metal layer,    -   b) contacting said metal layer with one or more molecules        according to claim 0 and with one or more molecules having a        formula Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂,    -   wherein said one or more molecules according to claim 0 and said        one or more molecules having a formula        Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂ self-assemble to form a mixed        layer of one or more first species and one or more second        species bound onto said metal layer, wherein each of said one or        more first species is a molecule having a structural formula        X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M and each of said one        or more second species is a molecule        Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M wherein:    -   Y₂ is either H or —S—R₄—(O—CH₂—[CH₂]_(t))_(n)—Y₁′,    -   R₁, R₃ and R₄ are each independently a saturated or        ethylenically unsaturated hydrocarbyl group with 3 to 30 carbon        atoms selected from the group consisting of alkyl, alkenyl,        cycloalkyl, cycloalkyl-alkyl, cycloalkenyl, cycloalkenylalkyl        and cycloalkylalkenyl, said group optionally comprising one or        more heteroatoms selected from nitrogen, oxygen and sulfur in        the main chain, and said group optionally comprising one or more        oxo substituents,    -   Y₁ and Y₁′ are independently either a hydroxy group or a methoxy        group,    -   M is said metal layer to which said one or more molecules are        bounded,    -   t is an integer from 1 to 2,    -   n is an integer from 3 to 15,000,    -   c is an integer from 0 to 3,    -   X₁ is selected from the group consisting of fluorophenyl,        fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl,        oxiranyl, aziridinyl, C₂₋₁₂ alkenyl, imino-ether,        dichlorotriazinyl, sulfonyl halide, alkoxycarbonyl,        isothiocyanato, isocyanato, carbonyl halide, haloalkylcarbonyl,        carboxylic acid anhydride, diazonium carbonyl,        N-(2-oxotetrahydro-3-thienyl)amido and        N-carboxy-thiazolidinyl-2-thione.

Preferably, a treatment to remove any metal oxide layer present at thesurface of the metal layer is performed according to any method wellknown by the person skilled in the art.

In a fifth aspect, a sensor is provided for the detection of an analytein a sample fluid, the device comprising:

-   -   a device according to the second aspect on which at least one        biomolecule is immobilized by a covalent bound, and    -   a transducer.

FIG. 3 shows the surface plasmon resonance (SPR) signal obtained forvarious concentrations of a prostate specific antigen (PSA) put inpresence of an anti-PSA antibody immobilized on a gold surface viacovalent bonding with a monolayer of species obtained from Example 8.The binding signals are expressed in resonance units (RU).

FIG. 4 shows comparative SPR data for the immobilization of anti-PSAantibody on [1] a self-assembled monolayer of the prior art activatedvia an activation step with a carbodiimide followed by apentafluorophenol binding and [2] on a self-assembled monolayeraccording to Example 8. The binding signal is expressed in resonanceunits (RU) and this signal is clearly higher in the case of theexemplary embodiment. In a sixth aspect, a method for manufacturing asensor according to the fifth aspect is provided, wherein the methodcomprises the steps of:

-   -   contacting a device according to the second aspect with a        solution of at least one biomolecule,    -   connecting said device to a transducer.

In an embodiment of this sixth aspect, the at least one biomolecule hasat least one primary amino group. In another embodiment of this sixthaspect, the device and/or said at least one biomolecule is notchemically activated prior to contacting.

Example 1 Synthesis of2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethyl2,3,4,5,6-pentafluorobenzoateand analogues derived from higher polyethylene glycols

2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethyl-2,3,4,5,6-pentafluorobenzoate is an example of a compound (shown schematically below) of thegeneral formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂)_(n)—O—R₁—S—X₂ whereinX₁=fluorobenzoyl, t=1, c=0 and n=3.

The present molecule has been synthesized according to the followingsynthesis scheme:

A solution of 2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3)(commercially available at Prochimia) in ethanol containing 4 molarequivalents of thioacetic acid and 10 mg of AIBN was irradiated under UVlight for 6 hours under an atmosphere of nitrogen. The reaction mixturewas concentrated by rotary evaporation at reduced pressure andpurification by chromatography on silica gel.

A mixture of NaH and the thioacetate in THF/DMF was stirred for about 1hour under an atmosphere of argon, and then pentafluorobenzoyl chloridewas added. After 36 hours, the reaction mixture was concentrated byrotary evaporation at reduced pressure and extracted withdichloromethane and water. The organic portion was dried over MgSO₄ andconcentrated by rotary evaporation at reduced pressure. The product waspurified by chromatography on silica gel.

A solution of the thioacetate in 0.1M HCl in MeOH was deprotected underan atmosphere of nitrogen by refluxing for about 4 h. The reactionmixture was concentrated by rotary evaporation at reduced pressure andpurification by chromatography on silica gel.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 2 Synthesis of2-(2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy)methylpentafluorophenoxycarbonyl (n=3) and analogues derived from higherpolyethylene glycols

2-(2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy)methylpentafluorophenoxycarbonyl is an example of a compound (shownschematically below) of the general formulaX₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂)_(n)—O—R₁—S—X₂ whereinX₁=pentafluorophenoxycarbonyl, c=1 and n=3.

The present molecule has been synthesized according to the followingsynthesis scheme:

NaH was added to a DMF/THF solution of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3) (commerciallyavailable from Prochimia). The mixture was stirred for about 10 min. andtert-butyl bromoacetate was added dropwise. The mixture was stirred atRT overnight and was concentrated by rotary evaporation at reducedpressure and extracted with ethyl acetate and water. The reactionmixture was concentrated by rotary evaporation at reduced pressure andpurification by chromatography on silica gel.

To a solution of the tert-butyl acetate derivative in toluene was addedthioacetic acid and AIBN. The mixture was irradiated under UV light for6 hours under an atmosphere of nitrogen. The reaction mixture wasconcentrated by rotary evaporation at reduced pressure and purificationby chromatography on silica gel.

A solution of the thioacetate in DMF was activated withdicyclohexyl-carbodiimide under an atmosphere of nitrogen by stirring atroom temperature for about 12 hours and then pentafluorophenol wasadded. The reaction mixture was stirred at room temperature andextracted with water. The reaction mixture was concentrated by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel.

A solution of the thioacetate in 0.1M HCl in MeOH was deprotected underan atmosphere of nitrogen by refluxing for about 4 hours. The reactionmixture was concentrated by rotary evaporation at reduced pressure andpurification by chromatography on silica gel.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 3 Synthesis of2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy chloridocarbonyl(n=3) and analogues derived from higher polyethylene glycols

2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy chloridocarbonyl isan example of a compound (shown schematically below) of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=carbonylchloride, c=0 and, X₂=H, R₁=(CH₂)₁₁.

A mixture of 1.5 molar equivalent NaH and 1 molar equivalent2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3) (commerciallyavailable at Prochimia) in 50 ml DMF was stirred for about 30 minutesunder an atmosphere of argon, and then 1.5 molar equivalent of carbonyldichloride was added. Next, the solution was stirred for 24 hours underan atmosphere of argon, followed by quenching with 25 ml methanol. Thereaction mixture was then extracted with diethyl ether and water. Theorganic portion was dried over MgSO₄ and concentrated by rotaryevaporation at reduced pressure. The product was purified bychromatography on silica gel.

A solution of this product (1 molar equivalent) in methanol containing 4molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) were irradiated under UV light for24 hours under an atmosphere of argon. The reaction mixture wasconcentrated by rotary evaporation at reduced pressure and purificationby chromatography on silica gel.

A solution of the thioacetate (1 molar equivalent) in a solutioncontaining 1M HCl and MeOH (1/1) was deprotected under an atmosphere ofnitrogen by stirring the reaction for 24 hours. The solution was thenextracted with an organic solvent and H₂O, followed by drying of theorganic phase on MgSO₄. The reaction mixture was concentrated by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel. Next, the product was again concentrated using rotaryevaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 4 Synthesis of1-chloro-3-(2-[2-(2-(11-mercaptoundecyloxy)-ethoxy)ethoxy]ethoxy)propan-2-oneand analogues derived from higher polyethylene glycols

1-chloro-3-(2-[2-(2-(11-mercaptoundecyloxy)-ethoxy)ethoxy]ethoxy)propan-2-one is an example of a compound (shown schematically below) ofthe general formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=chloroalkylcarbonyl, c=1 and n=3, t=1, X₂=H, R₁=(CH₂)₁₁.

A mixture of 1.5 molar equivalent NaH and 1 molar equivalent2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3) (commerciallyavailable at Prochimia) in 50 ml DMF was stirred for about 30 minutesunder an atmosphere of argon, and then 1.5 molar equivalent of1,3-dichloroacetone was added. Next, the solution was stirred for 24hours under an atmosphere of argon, followed by quenching with 25 mlmethanol. The reaction mixture was then extracted with diethyl ether andwater. The organic portion was dried over MgSO₄ and concentrated byrotary evaporation at reduced pressure. The product was purified bychromatography on silica gel.

A solution of this product (1 molar equivalent) in methanol containing 4molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) AIBN were irradiated under UV light for 24hours under an atmosphere of argon. The reaction mixture wasconcentrated by rotary evaporation at reduced pressure and purificationby chromatography on silica gel.

A solution of the thioacetate (1 molar equivalent) in a solutioncontaining 1M HCl and MeOH (1/1) was deprotected under an atmosphere ofnitrogen by stirring the reaction for 24 hours. The solution was thenextracted with an organic solvent and H₂O, followed by drying of theorganic phase on MgSO₄. The reaction mixture was concentrated by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel. Next, the product was again concentrated using rotaryevaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 5 Synthesis of11-(2-(2-(2-(oxiran-2-ylmethoxy)ethoxy)ethoxy)-ethoxy)undecane-1-thioland analogues derived from higher polyethylene glycols

11-(2-(2-(2-(oxiran-2-ylmethoxy)ethoxy)ethoxy)-ethoxy)undecane-1-thiolis an example of a compound (shown schematically below) of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=oxiranyl, c=1 and n=3, t=1, X₂=H, R₁=(CH₂)₁₁.

This molecule has been synthesized according to the following synthesisscheme:

A mixture of 1.5 molar equivalent NaH and 1 molar equivalent2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3) (commerciallyavailable at Prochimia) in 50 ml DMF was stirred for about 30 minutesunder an atmosphere of argon, and then 1.5 molar equivalent of2-(chloromethyl)oxirane was added. Next, the solution was stirred for 24hours under an atmosphere of argon, followed by quenching with 25 mlmethanol. The reaction mixture was then extracted with diethyl ether andwater. The organic portion was dried over MgSO₄ and concentrated byrotary evaporation at reduced pressure. The product was purified bychromatography on silica gel.

A solution of this product (1 molar equivalent) in methanol containing 4molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) were irradiated under UV light for24 hours under an atmosphere of argon. The reaction mixture wasconcentrated by rotary evaporation at reduced pressure and purificationby chromatography on silica gel.

A solution of the thioacetate (1 molar equivalent) in a solutioncontaining 1M HCl and MeOH (1/1) was deprotected under an atmosphere ofnitrogen by stirring the reaction for 24 hours. The solution was thenextracted with an organic solvent and H₂O, followed by drying of theorganic phase on MgSO₄. The reaction mixture was concentrated by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel. Next, the product was again concentrated using rotaryevaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 6 Synthesis of2-(2-(2-(11-mercaptoundecyloxy)ethoxy)-ethoxy)ethoxy acetylchloride andanalogues derived from higher polyethylene glycols

2-(2-(2-(11-mercaptoundecyloxy)ethoxy)-ethoxy)ethoxy acetylchloride isan example of a compound (shown schematically below) of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=carbonylchloride, c=1 and n=3, t=1, X₂=H, R₁=(CH₂)₁₁.

The present molecule has been synthesized according to the followingsynthesis scheme:

NaH (1.5 molar equivalent) was added to a 50 ml DMF solution of 1 molarequivalent of 2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol (n=3). Themixture was stirred for about 30 minutes and tert-butyl bromoacetate(1.5 molar equivalent) was added dropwise. The mixture was stirred atroom temperature for 24 hours under an atmosphere of argon. The productwas quenched with 25 ml of methanol. The reaction mixture was thenextracted with diethyl ether and water. The organic portion was driedover MgSO₄ and concentrated by rotary evaporation at reduced pressure.The product was purified by chromatography on silica gel. The resultingproduct was concentrated using rotary evaporation. The olefin wasdeprotected in a mixture of (1/1) HCl and H₂O under an atmosphere ofnitrogen. The reaction mixture was then concentrated by rotaryevaporation at reduced pressure and extracted with dichloromethane andwater. The organic portion was dried over MgSO₄ and concentrated byrotary evaporation at reduced pressure. The product was purified bychromatography on silica gel.

In an Erlenmeyer flask fitted by a ground-glass joint to a refluxcondenser capped with a calcium chloride drying tube,2-(2-(2-undec-10-enyloxy-ethoxy)-ethoxy)-acetic acid (n=3) (1 molarequivalent) and thionyl chloride (1.2 molar equivalents) were placed.The flask was warmed for 3 days in a heating bath kept at 45-50° C.Finally the mixture was heated at 60° C. for 5 hours. After cooling, itwas transferred to a modified Claisen flask and distilled at reducedpressure. A calcium chloride guard tube was inserted between the vacuumline and the apparatus, and the flask was heated with a bath.

A solution of the olefin (1 molar equivalent) in methanol containing 4molar equivalents of thioacetic acid and 10 mg of AIBN were irradiatedunder UV light for 24 hours under an atmosphere of argon. The productwas purified by filtering on a glass filter followed by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel.

A solution of the thioacetate (1 molar equivalent) in a solutioncontaining 1M HCl and MeOH (1/1) was deprotected under an atmosphere ofnitrogen by stirring the reaction for 24 hours. The solution was thenextracted with an organic solvent and H₂O, followed by drying of theorganic phase on MgSO₄. The reaction mixture was concentrated by rotaryevaporation at reduced pressure and purification by chromatography onsilica gel. Next, the product was again concentrated using rotaryevaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 7 Synthesis of2-{2-[2-(2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethyl2,3,4,5,6-pentafluoro-benzoate and analogues derived from higherpolyethylene glycols

2-{2-[2-(2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy)ethoxy]-ethoxy}ethyl2,3,4,5,6-pentafluoro-benzoate is an example of a compound of thegeneral formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=fluorobenzoyl, X₂=H, c=0, t=1, n=6, and R₁=(CH₂)₁₁. The presentmolecule has been synthesized according to the following synthesisscheme in three steps:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 0.0058 mole of2-[2-(2-{2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxyethanol was dissolved in a sufficient amount of ethanol. 0.023 mole ofthioacetic acid and 10 mg of 2,2′-azobis-isobutyronitrile (AIBN) wereadded and the solution was irradiated with UV light for 24 hours underan atmosphere of argon. The product was purified by filtering on a glassfilter followed by rotary evaporation at reduced pressure and bypurification on a silica column using ethyl acetate/methanol (95/5). Theproduct was obtained in 89% yield and was characterized as follows: ¹HNMR (300 MHz, CDCl₃): δ 3.65 (20H, m), δ 3.45 (2H, t), δ 2.85 (2H, t), δ2.5 (1H, s), δ 2.30 (3H, s), and 1.65-1.23 (18H, m).

In a second step, in a dry bottle having a magnetic stirrer and underargon atmosphere, 0.0052 mole of the product obtained in step 1 wasdissolved in a sufficient amount of CH₂Cl₂. 0.00785 mole oftriethylamine was added and the solution was stirred for 1 hour under anatmosphere of argon. 0.00785 mole of pentafluorobenzoyl chloride wasthen added and the solution was stirred for 24 hours under an atmosphereof argon. The product was purified by rotary evaporation at reducedpressure and by purification on a silica column using ethylacetate/methanol (95/5). The product was obtained in 80% yield and wascharacterized as follows: ¹H NMR (300 MHz, CDCl₃): δ 4.53 (2H, t), δ3.82 (2H, t), δ 3.65 (20H, m), δ 3.45 (2H, t), δ 2.85 (2H, t), δ 2.30(3H, s), and 1.65-1.23 (18H, m).

In a third step, in a dry bottle having a magnetic stirrer and underargon atmosphere, 0.00248 mole of the product obtained in step 2 wasdissolved in an excess amount of solution containing 1M HCl and EtOH(1/1). The solution was stirred for 24 hours. The solution was thenextracted with diethyl ether and H₂O. The organic phase was dried onMgSO₄. The product was then concentrated using rotary evaporation. Next,the product was purified on a silica column using ethyl acetate. Theproduct was again concentrated using rotary evaporation. The product wasobtained in 56% yield and was characterized as follows:

1H NMR (300 MHz, CDCl3): δ 4.53 (2H, t), δ 3.82 (2H, t), δ 3.65 (20H,m), δ 3.45 (2H, t), δ 2.51 (2H, q), 1.60-1.27 (18H, m), and

C13 NMR (300 MHz, CDCl₃): 159.27; 148.61; 143.98; 139.89; 136.54;108.31; 70.93; 70.38; 69.0; 66.09; 34.38; 29.96; 29.81; 29.38; 28.68;26.42; and 24.93 ppm.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-[2-(2-{2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxyethanol. If necessary, type and/or amount of solvent and/or reactiontime may be adapted in view of n, especially accounting for the physicalstate (liquid or solid) and the solubility of the polyethyleneglycolinvolved.

Example 8 Synthesis of2-(2-{2-[2-(2-{2-[2-(11-mercaptoyldisulfanyl-undecyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}aceticacid pentafluorophenyl ester and analogues derived from higherpolyethylene glycols

2-(2-{2-[2-(2-{2-[2-(11-mercapto-undecyloxy)ethoxy]ethoxy}ethoxy)ethoxy]-ethoxy}ethoxy)methylpentafluorophenoxycarbonyl is an example of a compound of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=pentafluorophenoxycarbonyl,X₂=X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S, c=1, t=1, n=6, andR₁=(CH₂)₁₁, which has been synthesized according to the followingscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 9.2 mmole of2-[2-(2-{2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxyethanol and 13.8 mmole of NaH were dissolved in 50 mL DMF. The solutionwas stirred for 30 minutes. 13.8 mmole of methyl bromo-acetate was addedand the solution was stirred for 24 hours under an atmosphere of argon.The product was quenched with 25 mL of methanol. The solution was thenextracted with diethyl ether and H₂O. The organic phase was dried onMgSO₄. The product was then concentrated using rotary evaporation. Next,the product was purified on a silica column using ethylacetate. Theproduct was again concentrated using rotary evaporation. The product wasobtained in 50% yield and was characterized as follows: 1H NMR (300 MHz,CDCl₃): δ 5.82 (1H, m), δ 4.98 (2H, m), δ 4.18 (2H, s), δ 3.75 (3H, s),δ 3.65 (24H, m), δ 3.41 (2H, t), and 1.65-1.23 (18H, m).

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1.2 mmole of the product obtained in step 2 wasdissolved in a sufficient amount of methanol. 4.6 mmole of thioaceticacid and 10 mg of 2,2′-azobis-isobutyronitrile (AIBN) were added and thesolution was irradiated with UV light for 24 hours under an atmosphereof argon. The product was purified by filtering on a glass filterfollowed by rotary evaporation at reduced pressure and by purificationon a silica column using ethyl acetate/methanol (95/5). The product wasobtained in 83% yield and was characterized as follows: 1H NMR (300 MHz,CDCl₃): δ 4.18 (2H, s), δ 3.75 (3H, s), δ 3.65 (22H, m), δ 3.45 (2H, t),δ 2.85 (2H, t), δ 2.33 (3H, s), and 1.65-1.23 (18H, m).

In a third step, in a dry bottle comprising a magnetic stirrer, 1 molarequivalent (0.2 g) of the product obtained in step 2 was dissolved in asolution containing methanol/1M NaOH (3/1) (15 mL/5 mL). The solutionwas stirred for 24 hours. HCl was added to acidify the solution. Thesolution was then extracted with CH₂Cl₂ and H₂O. The organic phase wasdried on MgSO4. The product is then concentrated using rotaryevaporation. Yield: 98%. 1H NMR (300 MHz, CDCl₃): δ 4.18 (4H, s), δ 3.65(41H, m), δ 3.45 (4H, t), δ 2.65 (4H, t), 1.65-1.23 (36H, m).

In a fourth step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 9.5 10⁻⁵ mole of the product obtained in step 3and 1.14 10⁻⁴ mole pentafluorophenol (PFP) was dissolved in dry ethylacetate (100 mL). The solution was stirred and cooled down to 0° C. 1.1410⁻⁴ mole dicyclohexyl-carbodiimide (DCC) was added and the solution wasstirred for 30 minutes. Next, the solution was stirred for 3 days atroom temperature. The product was concentrated by rotary evaporation atreduced pressure. The product was obtained in 84% yield and wascharacterized as follows:

1H NMR (300 MHz, CDCl3): δ 4.56 (4H, s), δ 4.5-3.73 (48H, m), δ 3.45(4H, t), δ 2.69 (4H, t), δ 1.75-1.28 (58H, m) (36H, m+22H),

C13 (300 MHz, CDCl3): 166.61; 157.13; 142.27; 139.18; 136.66; 131.95;71.53; 70.58; 68.97; 67.87; 49.32; 39.21; 33.81; 29.48; 29.22; 28.52;26.06; 25.53; and 24.83 ppm; and

MS: 1405.5 (M+).

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolderivative is used instead of2-[2-(2-{2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxyethanol. If necessary, type and/or amount of solvent and/or reactiontime may be adapted in view of n, especially accounting for the physicalstate (liquid or solid) and the solubility of the polyethyleneglycolinvolved.

Example 9 Synthesis of2-(2-(2-(2-(11-2-(allyloxy)ethoxy)ethoxy)ethoxy)ethoxyundec-6-ene-1-thiol and analogues derived from higher polyethyleneglycols

2-(2-(2-(2-(11-2-(allyloxy)ethoxy)ethoxy)ethoxy)ethoxyundec-6-ene-1-thiol is an example of a compound (shown schematicallybelow) of the general formulaX₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=alkenyl, c=1,t=1 and X₂=H.

This molecule and is synthesized according to the following scheme:

In a first step, in a dry bottle comprising a magnetic stirrer, a ballcooler and under argon atmosphere, 1 molar equivalent of5,10-undecadien-1-ol (product # S303453-1 EA SigmaAldrich) is dissolvedin dry dichloromethane.

The bottle is cooled in a mixture of acetone and dry CO₂ (−23° C.). 1molar equivalent of triphenylphosphine and 1 molar equivalent ofN-bromosuccinimide are added. The reaction mixture is stirred for 1 h ata temperature of −23° C. and afterwards for ½ h at room temperature. Thesolution is extracted with a solution of sodium carbonate. The organicphase is dried on MgSO₄. The product is then concentrated using rotaryevaporation. Next, the product is extracted with hexane under reflux.The product is filtered via vacuum filtration on alumina (5 cm in heightand 3 cm diameter) and washed with hexane. The product is againconcentrated using rotary evaporation.

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 2 molar equivalents of triethylene glycol aredissolved in a mixture of THF/DMF. 1 molar equivalent of NaH is addedand the solution is stirred for 1 h under an atmosphere of argon. 1molar equivalent of the product obtained in step 1 is added, and thereaction mixture is stirred for 3 days under an atmosphere of argon.

The product is quenched with MeOH. THF and DMF are removed by rotaryevaporation at reduced pressure. The product is extracted withdichloromethane and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known by persons skilled in the art.

The product is again concentrated using rotary evaporation.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in Ethanol. 4 molar equivalents of thioacetic acid and 10mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated for 24 h under an atmosphere of argon. The product ispurified by filtering on a glass filter followed by rotary evaporationat reduced pressure and by purification on a silica column usingethylacetate/methanol (95/5).

In a fourth step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 3, is dissolved in CH₂Cl₂. 1.5 molar equivalents of triethylamineis added and the solution is stirred for 1 h under an atmosphere ofargon. 1.5 molar equivalents of allyl bromide are then added and thesolution is stirred for 24 h under an atmosphere of argon. The productis purified by rotary evaporation at reduced pressure and bypurification on a silica column using a suitable mixture of organicsolvents, known by persons skilled in the art.

In a fifth step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 4is dissolved in a solution containing 1M HCl and MeOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known by persons skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolis used instead of triethylene glycol. If necessary, type and/or amountof solvent and/or reaction time may be adapted in view of n, especiallyaccounting for the physical state (liquid or solid) and the solubilityof the polyethyleneglycol involved.

Example 10 Synthesis of 2-(2-(2-(allyloxy)ethoxy)ethoxy)ethyl16-mercapto-6-oxohexadecanoate and analogues derived from higherpolyethylene glycols

2-(2-(2-(allyloxy)ethoxy)ethoxy)ethyl 16-mercapto-6-oxohexadecanoate isan example of a compound (shown schematically below) of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=alkenyl,c=1, t=1, X₂=H and n=3.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of 6-oxo-15-hexadecenoic acid and 2molar equivalents of triethylene glycol are dissolved in dryethylacetate. The solution is stirred and cooled down to 0° C. 1.2 molarequivalents dicyclohexyl-carbodiimide (DCC) is added and the solution isstirred for 30 min. Next, the solution is stirred for 3 days at roomtemperature. The product is purified on a silica column using a suitablemixture of organic solvents, known by the person skilled in the art. Theresulting product is concentrated using rotary evaporation

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1 is dissolved in Ethanol. 4 molar equivalents of thioacetic acidand 10 mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and thesolution is irradiated for 24 h under an atmosphere of argon. Theproduct is purified by filtering on a glass filter followed by rotaryevaporation at reduced pressure and by purification on a silica columnusing ethylacetate/methanol (95/5).

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2,is dissolved in CH₂Cl₂. 1.5 molar equivalents of triethylamine is addedand the solution is stirred for 1 h under an atmosphere of argon. 1.5molar equivalents of allyl bromide are then added and the solution isstirred for 24 h under an atmosphere of argon. The product is purifiedby rotary evaporation at reduced pressure and by purification on asilica column using a suitable mixture of organic solvents, known by theperson skilled in the art.

In a fourth step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 3 is dissolved in a solution containing 1M HCl and MeOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known by persons skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethylene glycolis used instead of triethylene glycol. If necessary, type and/or amountof solvent and/or reaction time may be adapted in view of n, especiallyaccounting for the physical state (liquid or solid) and the solubilityof the polyethyleneglycol involved.

Example 11 Synthesis of2-(2-{2-[2-(11-mercaptoyldisulfanyl-undecyloxy)ethoxy]ethoxy}ethoxy)methyl2,4-dinitrophenoxycarbonyl

2-(2-{2-[2-(11-mercaptoyldisulfanyl-undecyloxy)ethoxy]ethoxy}ethoxy)methyl2,4-dinitrophenoxycarbonyl is an example of a compound (shownschematically below) of the general formulaX₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=2,4-dinitrophenoxycarbonyl, c=1 and n=3, t=1,X₂=X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S, and R₁=(CH₂)₁₁.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol and 1.5 molarequivalents of NaH are dissolved in 50 mL DMF. The solution is stirredfor 30 min. 1.5 molar equivalents of methyl bromoacetate is added andthe solution is stirred for 24 h under an atmosphere of argon. Theproduct is quenched with 25 mL of methanol. The solution is thenextracted with diethyl ether and H₂O. The organic phase is dried onMgSO₄. The product is then concentrated using rotary evaporation. Next,the product is purified on a silica column using a suitable mixture oforganic solvents, known to the person skilled in the art. The resultingproduct is concentrated using rotary evaporation.

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1 is dissolved in methanol. 4 molar equivalents of thioacetic acidand 10 mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and thesolution is irradiated under UV light for 24 h under an atmosphere ofargon. The product is purified by filtering on a glass filter followedby rotary evaporation at reduced pressure and by purification on asilica column using a suitable mixture of organic solvents, known to theperson skilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and 1molar equivalent of the product obtained in step 2 is dissolved in asolution containing methanol/1M NaOH (3/1). The solution is stirred for24 h. HCl is added to acidify the solution. The solution is thenextracted with CH₂Cl₂ and H₂O. The organic phase is dried on MgSO₄. Theproduct is purified on a silica column using a suitable mixture oforganic solvents, known to the person skilled in the art. The resultingproduct is concentrated using rotary evaporation.

In a fourth step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 3 and 1.2 molar equivalent phenol is dissolved in dry ethylacetate.The solution is stirred and cooled down to 0° C. 1.2 molar equivalentsdicyclohexyl-carbodiimide (DCC) is added and the solution is stirred for30 min. Next, the solution is stirred for 3 days at room temperature.The product is purified on a silica column using a suitable mixture oforganic solvents, known to the person skilled in the art. The resultingproduct is concentrated using rotary evaporation.

In a fifth step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 4and 2 molar equivalents HNO₃ in H₂SO₄ is added. The solution is stirredfor 24 h. The solution is then extracted with organic solvents and H₂O.The organic phase is dried on MgSO₄. The product is purified on a silicacolumn using a suitable mixture of organic solvents, known to the personskilled in the art. The resulting product is concentrated using rotaryevaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 12 Synthesis of2-{2-[2-(11-mercapto-undecyloxy)-ethoxy]-ethoxy}-ethoxy propene andanalogues derived from higher polyethylene glycols

2-{2-[2-(11-mercapto-undecyloxy)-ethoxy]-ethoxy}-ethoxy propene is anexample of a compound (shown schematically below) of the general formulaX₁—(CH₂)_(n)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=alkenyl, c=1,t=1, X₂=H, and R₁=(CH₂)₁₁, and n=3.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol is dissolved in Ethanol.4 molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated for 24 h under an atmosphere of argon. The product ispurified by filtering on a glass filter followed by rotary evaporationat reduced pressure and by purification on a silica column usingethylacetate/methanol (95/5).

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1, is dissolved in CH₂Cl₂. 1.5 molar equivalents of triethylamineis added and the solution is stirred for 1 h under an atmosphere ofargon. 1.5 molar equivalents of allyl bromide are then added and thesolution is stirred for 24 h under an atmosphere of argon. The productis purified by rotary evaporation at reduced pressure and bypurification on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and MeOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 13 Synthesis of Synthesis of2-(2-(2-(11-mercapto-undecyloxy)-ethoxy)ethoxy)ethoxy sulfonylchlorideand analogues derived from higher polyethylene glycols.

2-(2-(2-(11-mercapto-undecyloxy)ethoxy)ethoxy)ethoxy sulfonyl chlorideis an example of a compound (shown schematically below) of the generalformula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₁=sulfonylchloride, c=0 and n=3, t=1, X₂=H, and R₁=(CH₂)₁₁.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol is dissolved in ethanol.4 molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated for 24 h under an atmosphere of argon. The product ispurified by filtering on a glass filter followed by rotary evaporationat reduced pressure and by purification on a silica column usingethylacetate/methanol (95/5).

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1, is dissolved in an organic solvent. 1.5 molar equivalents oftriethylamine are added and the solution is stirred for 1 h under anatmosphere of argon. 1.5 molar equivalents of sulfuryl chloride fluorideare then added and the solution is stirred for 24 h under an atmosphereof argon. The product is purified by rotary evaporation at reducedpressure and by purification on a silica column using a suitable mixtureof organic solvents, known by the person skilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and MeOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 14 Synthesis of2-(2-(2-(11-mercaptoyldisulfanyl-undecyloxy)-ethoxy)ethoxy)ethoxysulfonylchloride and analogues derived from higher polyethylene glycols

2-(2-(2-(11-mercaptoyldisulfanyl-undecyloxy)-ethoxy)ethoxy)ethoxysulfonylchloride is an example of a compound (shown schematically below)of the general formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂wherein X₁=sulfonyl chloride, c=0 and n=3, t=1,X₂=X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S, and R₁=(CH₂)₁₁.

This molecule is synthesized according to the following scheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol is dissolved in Ethanol.4 molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated under UV for 24 h under an atmosphere of argon. The productis purified by filtering on a glass filter followed by rotaryevaporation at reduced pressure and by purification on a silica columnusing ethylacetate/methanol (95/5).

In a second step, in a dry bottle comprising a magnetic stirrer and 1molar equivalent of the product obtained in step 1 is dissolved in asolution containing methanol/1M NaOH (3/1). The solution is stirred for24 h. The solution is then extracted with an organic solvent and H₂O.The organic phase is dried on MgSO₄. The product is purified on a silicacolumn using a suitable mixture of organic solvents, known the personskilled in the art. The resulting product is concentrated using rotaryevaporation.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2,is dissolved in an organic solvent. 1.5 molar equivalents oftriethylamine are added and the solution is stirred for 1 h under anatmosphere of argon. 1.5 molar equivalents of sulfuryl chloride fluorideare then added and the solution is stirred for 24 h under an atmosphereof argon. The product is purified by rotary evaporation at reducedpressure and by purification on a silica column using a suitable mixtureof organic solvents, known to the person skilled in the art.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 15 Synthesis of2-(2-(2-(2-(11-mercaptoyldisulfanyl-undecyloxy)-ethoxy)ethoxy)ethoxy)methylisothiocyanate and analogues derived from higher polyethylene glycols

2-(2-(2-(2-(11-mercaptoyldisulfanyl-undecyloxy)-ethoxy)ethoxy)ethoxy)methylisothiocyanate is an example of a compound (shown schematically below)of the general formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂wherein X₁=isothiocyanato, c=1 and n=3, t=1,X₂=X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S, and R₁=(CH₂)₁₁.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol is dissolved in Ethanol.4 molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated for 24 h under an atmosphere of argon. The product ispurified by filtering on a glass filter followed by rotary evaporationat reduced pressure and by purification on a silica column usingethylacetate/methanol (95/5).

In a second step, in a dry bottle comprising a magnetic stirrer and 1molar equivalent of the product obtained in step 1 is dissolved in asolution containing methanol/1M NaOH (3/1). The solution is stirred for24 h. The solution is then extracted with an organic solvent and H₂O.The organic phase is dried on MgSO₄. The product is purified on a silicacolumn using a suitable mixture of organic solvents, known to the personskilled in the art. The resulting product is concentrated using rotaryevaporation.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2,is dissolved in an organic solvent. 1.5 molar equivalents oftriethylamine are added and the solution is stirred for 1 h under anatmosphere of argon. 1.5 molar equivalents of chloromethyl thiocyanateare then added and the solution is stirred for 24 h under an atmosphereof argon. The product is purified by rotary evaporation at reducedpressure and by purification on a silica column using a suitable mixtureof organic solvents, known to the person skilled in the art.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 16 Synthesis of2-(2-(2-(2-(11-mercapto-undecyloxy)-ethoxy)ethoxy)ethoxy)methylisothiocyanate and analogues derived from higher polyethylene glycols

2-(2-(2-(2-(11-mercapto-undecyloxy)ethoxy)ethoxy)ethoxy)methylisothiocyanate is an example of a compound (shown schematically below)of the general formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂wherein X₁=isothiocyanato, c=1, t=1, X₂=H, and R₁=(CH₂)₁₁, and n=3.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol is dissolved in Ethanol.4 molar equivalents of thioacetic acid and 10 mg of2,2′-azobis(isobutyronitrile) (AIBN) are added and the solution isirradiated for 24 h under an atmosphere of argon. The product ispurified by filtering on a glass filter followed by rotary evaporationat reduced pressure and by purification on a silica column usingethylacetate/methanol (95/5).

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1, is dissolved in CH₂Cl₂. 1.5 molar equivalents of triethylamineis added and the solution is stirred for 1 h under an atmosphere ofargon. 1.5 molar equivalents of chloromethyl thiocyanate are then addedand the solution is stirred for 24 h under an atmosphere of argon. Theproduct is purified by rotary evaporation at reduced pressure and bypurification on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and MeOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation

Next, the product is purified on a silica column using a suitablemixture of organic solvents, known to the person skilled in the art. Theproduct is again concentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 17 Synthesis of2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyl1,3-chloropropanone and analogues derived from higher polyethyleneglycols.

2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyl1,3-chloropropanone is an example of a compound (shown schematicallybelow) of the general formulaX₁—(CH₂)_(n)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ whereinX₁=chloroalkylcarbonyl, c=1, t=1, X₂=H, and R₁=(CH₂)₁₁, and n=3.

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol and 1.5 molarequivalents of NaH are dissolved in 50 mL DMF. The solution is stirredfor 30 min. 1.5 molar equivalents of 3-chloropropionyl chloride is addedand the solution is stirred for 24 h under an atmosphere of argon. Theproduct is quenched with 25 mL of methanol. The solution is thenextracted with an organic solvent and H₂O. The organic phase is dried onMgSO₄. The product is then concentrated using rotary evaporation. Next,the product is purified on a silica column using a suitable mixture oforganic solvents, known to the person skilled in the art. The resultingproduct is concentrated using rotary evaporation.

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1 is dissolved in methanol. 4 molar equivalents of thioacetic acidand 10 mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and thesolution is irradiated under UV light for 24 h under an atmosphere ofargon. The product is purified by filtering on a glass filter followedby rotary evaporation at reduced pressure and by purification on asilica column using a suitable mixture of organic solvents, known to theperson skilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and EtOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation.

Next, the product is purified on a silica column using a suitablemixture of organic solvents, known to the person skilled in the art. Theproduct is again concentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 18 Synthesis of2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)-ethoxy)ethoxy)methyl4-fluorophenyl and analogues derived from higher polyethylene glycols

2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyl4-fluorophenyl is an example of a compound (shown schematically below)of the general formula X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂wherein X₁=fluorophenyl, c=1, t=1, X₂=H, and R₁=(CH₂)₁₁, and n=3:

The present molecule is synthesized according to the following synthesisscheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol and 1.5 molarequivalents of NaH are dissolved in 50 mL DMF. The solution is stirredfor 30 min. 1.5 molar equivalents of 4-fluorobenzylchloride is added andthe solution is stirred for 24 h under an atmosphere of argon. Theproduct is quenched with 25 mL of methanol. The solution is thenextracted with an organic solvent and H₂O. The organic phase is dried onMgSO₄. The product is then concentrated using rotary evaporation. Next,the product is purified on a silica column using a suitable mixture oforganic solvents, known to the person skilled in the art. The resultingproduct is concentrated using rotary evaporation.

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1 is dissolved in methanol. 4 molar equivalents of thioacetic acidand 10 mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and thesolution is irradiated for 24 h under an atmosphere of argon. Theproduct is purified by filtering on a glass filter followed by rotaryevaporation at reduced pressure and by purification on a silica columnusing a suitable mixture of organic solvents, known to the personskilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and EtOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 19 Synthesis of2,4-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyl-difluorophenyland analogues derived from higher polyethylene glycols

The same procedure as described for Example 18 is followed but thestarting material 4-fluorobenzylchloride is replaced byalpha-bromo-2,4-difluorotoluene.

Example 20 Synthesis of Synthesis of3,4-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyldifluorophenyl and analogues derived from higher polyethylene glycols

The same procedure as described for Example 18 is followed but thestarting material 4-fluorobenzylchloride is replaced byalpha-bromo-3,4-difluorotoluene.

Example 21 Synthesis of2,3,4,5-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methyl-tetrafluorophenyland analogues derived from higher polyethylene glycols

The same procedure as described for Example 18 is followed but thestarting material 4-fluorobenzylchloride is replaced by2,3,4,5-tetrafluorobenzylbromide

Example 22 Synthesis of2,3,4,5,6-(2-(2-(2-(2-(11-mercaptoundecyloxy)-ethoxy)ethoxy)ethoxy)methyl-pentafluorophenyland analogues derived from higher polyethylene glycols

2,3,4,5,6-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)methylpentafluorophenyl is an example of a compound (shown schematicallybelow) of the general formulaX₁—(CH₂)_(c)—(O—[CH₂]_(t)—CH₂)_(n)—O—R₁—S—X₂ whereinX₁=pentafluorophenyl, c=1, t=1, X₂=H, and R₁=(CH₂)₁₁, and n=3.

The present molecule can be synthesized according to the followingsynthesis scheme:

In a first step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol and 1.5 molarequivalents of NaH are dissolved in 50 mL DMF. The solution is stirredfor 30 min. 1.5 molar equivalents ofalpha-bromo-2,3,4,5,6-pentafluorotoluene is added and the solution isstirred for 24 h under an atmosphere of argon. The product is quenchedwith 25 mL of methanol. The solution is then extracted with an organicsolvent and H₂O. The organic phase is dried on MgSO₄. The product isthen concentrated using rotary evaporation. Next, the product ispurified on a silica column using a suitable mixture of organicsolvents, known by persons skilled in the art. The resulting product isconcentrated using rotary evaporation.

In a second step, in a dry bottle comprising a magnetic stirrer andunder argon atmosphere, 1 molar equivalent of the product obtained instep 1 is dissolved in methanol. 4 molar equivalents of thioacetic acidand 10 mg of 2,2′-azobis(isobutyronitrile) (AIBN) are added and thesolution is irradiated for 24 h under an atmosphere of argon. Theproduct is purified by filtering on a glass filter followed by rotaryevaporation at reduced pressure and by purification on a silica columnusing a suitable mixture of organic solvents, known to the personskilled in the art.

In a third step, in a dry bottle comprising a magnetic stirrer and underargon atmosphere, 1 molar equivalent of the product obtained in step 2is dissolved in a solution containing 1M HCl and EtOH (1/1). Thesolution is stirred for 24 h. The solution is then extracted with anorganic solvent and H₂O. The organic phase is dried on MgSO₄. Theproduct is then concentrated using rotary evaporation. Next, the productis purified on a silica column using a suitable mixture of organicsolvents, known to the person skilled in the art. The product is againconcentrated using rotary evaporation.

The above synthetic procedure can be executed with n taking any valuefrom 3 to 15,000 provided that a suitable oligo- or poly-ethyleneglycolderivative is used instead of2-{2-[2-(undec-10-enyloxy)ethoxy]ethoxy}ethanol. If necessary, typeand/or amount of solvent and/or reaction time may be adapted in view ofn, especially accounting for the physical state (liquid or solid) andthe solubility of the polyethyleneglycol involved.

Example 23 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-4-fluorobenzoate and analoguesderived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 4-fluorobenzoylchloride.

Example 24 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl 3,4-difluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 3,4-difluorobenzoylchloride.

Example 25 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-2,4-difluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 2,4-difluorobenzoylchloride.

Example 26 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-3,4,5-trifluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 3,4,5-trifluorobenzoylchloride.

Example 27 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-2,3,4-trifluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 2,3,4-trifluorobenzoylchloride.

Example 28 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-2,4,5-trifluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 2,4,5-trifluorobenzoylchloride.

Example 29 Synthesis of 2(-2(-2-(11mercaptoundecyloxy)ethoxy)ethoxy)ethyl-2,3,4,5-tetrafluorobenzoate andanalogues derived from higher polyethylene glycols

The same procedure as described for Example 1 is followed but thestarting material pentafluorobenzoyl chloride in the second step isreplaced by 2,3,4,5 tetrafluorobenzoylchloride.

Example 30 Method for Preparing a Device According to the Third Aspect

In the third aspect, the step of contacting the metal layer with one ormore molecules according to the first aspect can for instance beperformed in the following way:

The metal substrate such as gold is cleaned using UV/O₃ treatment. Themolecules can be deposited from a water-free organic solvent like forexample tetrahydrofuran (THF). The metal substrates are deposited inthis (for example 1 mM) solution and the optimal time (at least 3 h) isused to organize the thiols into a self-assembled monolayer (SAM).Afterwards the substrate with the SAM is rinsed with THF and dried withnitrogen. Next step is putting this substrate in a solution with therecognition molecules. The recognition molecules will covalently bindwithout any activation step.

Example 31 Method for Preparing a Device According to the Third Aspect

The metal substrate such as gold is cleaned using UV/O₃ treatment. Themolecules can be deposited from a water-free organic solvent like forexample tetrahydrofuran (THF). The metal substrates are deposited inthis (for example 1 mM) solution containing a mixture of moleculesaccording to the first aspect and molecules of the formulaY₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂ in chosen proportions. The optimal time(at least 3 h) is used to organize the thiols into a self-assembledmonolayer (SAM). Afterwards the substrate with the SAM is rinsed withTHF and dried with nitrogen. Next step is putting this substrate in asolution with the recognition molecules. The recognition molecules willcovalently bind without any activation step.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained. At the veryleast, and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldbe construed in light of the number of significant digits and ordinaryrounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention as embodied in the attached claims.

1. A device configured for immobilizing at least one biomolecule througha covalent bond, comprising: a substrate comprising a metal layer; atleast one first species bound to the metal layer, wherein each firstspecies is independently a molecule having a structural formula:X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M wherein: M is the metallayer to which the first species is bound; t is an integer from 1 to 2;n is an integer from 3 to 15,000; c is an integer from 0 to 3; R₁ is asaturated or ethylenically unsaturated hydrocarbyl group having from 3to 30 carbon atoms, wherein the hydrocarbyl group is selected from thegroup consisting of alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl,cycloalkenyl, cycloalkenylalkyl, and cycloalkylalkenyl, wherein thehydrocarbyl group is unsubstituted or substituted with at least onesubstituent selected from the group consisting of a heteroatom in themain chain and an oxo substituent, wherein the heteroatom is selectedfrom the group consisting of nitrogen, oxygen and sulfur; and X₁ isselected from the group consisting of fluorophenyl, fluorobenzoyl,fluorophenoxycarbonyl, nitrophenoxycarbonyl, oxiranyl, aziridinyl, C₂₋₁₂alkenyl, imino-ether, dichlorotriazinyl, sulfonyl halide,alkoxycarbonyl, isothiocyanato, isocyanato, carbonyl halide,haloalkylcarbonyl, carboxylic acid anhydride, diazonium carbonyl,N-(2-oxotetrahydro-3-thienyl)amido, andN-carboxy-thiazolidinyl-2-thione.
 2. The device of claim 1, wherein themetal layer comprises at least one metal selected from the groupconsisting of gold, silver, mercury, aluminum, platinum, palladium,copper, cadmium, lead, iron, chromium, manganese, tungsten, and alloysthereof.
 3. The device of claim 1, wherein the biomolecule has at leastone primary amino group, and wherein the biomolecule is immobilized byreaction of the primary amino group with the first species.
 4. Thedevice of claim 1, wherein the first species forms a monolayer on themetal layer.
 5. The device of claim 1, further comprising a transducerand at least one biomolecule immobilized by a covalent bond to thedevice, wherein the device is configured for use as a sensor for thedetection of an analyte in a sample fluid.
 6. The device of claim 1,further comprising at least one second species bound to the metal layer,wherein each second species is independently a molecule having astructural formulaY₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M wherein: R₃ is a saturated orethylenically unsaturated hydrocarbyl group having from 3 to 30 carbonatoms, wherein the hydrocarbyl group is selected from the groupconsisting of alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl,cycloalkenyl, cycloalkenylalkyl, and cycloalkylalkenyl, wherein thehydrocarbyl group is unsubstituted or substituted with at least onesubstituent selected from the group consisting of a heteroatom in themain chain and an oxo substituent, wherein the heteroatom is selectedfrom the group consisting of nitrogen, oxygen and sulfur; t is aninteger from 1 to 2; n is an integer from 3 to 15,000; M is said metallayer to which said one or more molecules are bounded; and Y₁ isselected from the group consisting of hydroxy and methoxy.
 7. The deviceof claim 6, wherein the first species and the second species form amixed monolayer on the metal layer.
 8. A process for preparing a deviceaccording to claim 1, comprising the steps of: providing a substratecomprising a metal layer; contacting the metal layer with at least onemolecule having a structural formula:X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S—X₂ wherein X₂ is selected fromthe group consisting of H and S—R₅; R₅ is an organic spacer selectedfrom the group consisting of R₂—(O—CH₂—[CH₂]_(t))_(n)—O—(CH₂)_(c)—X₁′and R₃—(O—CH₂—[CH₂]_(t))_(n)—Y₁, t is 1 or 2; c is an integer from 0 to3; n is an integer from 3 to 15,000; R₁, R₂ and R₃ are eachindependently a saturated or ethylenically unsaturated hydrocarbyl grouphaving from 3 to 30 carbon atoms, wherein the hydrocarbyl group isindependently selected from the group consisting of alkyl, alkenyl,cycloalkyl, cycloalkyl-alkyl, cycloalkenyl, cycloalkenylalkyl, andcycloalkylalkenyl, wherein the hydrocarbyl group is independentlyunsubstituted or substituted with at least one substituent selected fromthe group consisting of a heteroatom in the main chain and an oxosubstituent, wherein the heteroatom is independently selected from thegroup consisting of nitrogen, oxygen and sulfur; Y₁ is selected from thegroup consisting of hydroxy and methoxy; and X₁ and X₁′ are eachindependently selected from the group consisting of fluorophenyl,fluorobenzoyl, fluorophenoxycarbonyl, nitrophenoxycarbonyl, oxiranyl,aziridinyl, C₂₋₁₂ alkenyl, imino-ether, dichlorotriazinyl, sulfonylhalide, alkoxycarbonyl, isothiocyanato, isocyanato, carbonyl halide,haloalkylcarbonyl, carboxylic acid anhydride, diazonium carbonyl,N-(2-oxotetrahydro-3-thienyl)amido, andN-carboxy-thiazolidinyl-2-thione; whereby a self-assembled monolayerbound onto the metal layer, the self-assembled monolayer comprising atleast one first species bound to the metal layer, wherein each firstspecies is independently a molecule having a structural formula:X₁—(CH₂)_(c)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M wherein M is the metal layerto which the first species is bound; t is an integer from 1 to 2; n isan integer from 3 to 15,000; c is an integer from 0 to 3; R₁ is asaturated or ethylenically unsaturated hydrocarbyl group having from 3to 30 carbon atoms, wherein the hydrocarbyl group is selected from thegroup consisting of alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl,cycloalkenyl, cycloalkenylalkyl, and cycloalkylalkenyl, wherein thehydrocarbyl group is unsubstituted or substituted with at least onesubstituent selected from the group consisting of a heteroatom in themain chain and an oxo substituent, wherein the heteroatom is selectedfrom the group consisting of nitrogen, oxygen and sulfur; and X₁ isselected from the group consisting of fluorophenyl, fluorobenzoyl,fluorophenoxycarbonyl, nitrophenoxycarbonyl, oxiranyl, aziridinyl, C₂₋₁₂alkenyl, imino-ether, dichlorotriazinyl, sulfonyl halide,alkoxycarbonyl, isothiocyanato, isocyanato, carbonyl halide,haloalkylcarbonyl, carboxylic acid anhydride, diazonium carbonyl,N-(2-oxotetrahydro-3-thienyl)amido, andN-carboxy-thiazolidinyl-2-thione, whereby a device configured forimmobilizing at least one biomolecule through a covalent bond isobtained.
 9. The process of claim 8, further comprising: contacting thedevice with a solution of at least one biomolecule; and connecting thedevice to a transducer, whereby the device is configured for use as asensor for the detection of an analyte in a sample fluid.
 10. Theprocess of claim 9, wherein the biomolecule has at least one primaryamino group.
 11. The process of claim 9, wherein at least one of thedevice and the biomolecule is not chemically activated prior to the stepof contacting.
 12. The process of claim 8, further comprising:contacting the metal layer with at least one second molecule, whereineach second molecule independently has a structural formula:Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S—Y₂, wherein R₃ is a saturated orethylenically unsaturated hydrocarbyl group having from 3 to 30 carbonatoms, wherein the hydrocarbyl group is selected from the groupconsisting of alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl,cycloalkenyl, cycloalkenylalkyl, and cycloalkylalkenyl, wherein thehydrocarbyl group is unsubstituted or substituted with at least onesubstituent selected from the group consisting of a heteroatom in themain chain and an oxo substituent, wherein the heteroatom is selectedfrom the group consisting of nitrogen, oxygen and sulfur; t is aninteger from 1 to 2; n is an integer from 3 to 15,000; M is said metallayer to which said one or more molecules are bounded; and Y₁ isselected from the group consisting of hydroxy and methoxy; wherein thefirst molecule and the second molecule self-assemble to form a mixedmonolayer layer of at least one first species and at least one secondspecies bound to the metal layer, wherein each first species isindependently a molecule having a structural formulaX₁—(CH₂)_(n)—O—([CH₂]_(t)—CH₂—O)_(n)—R₁—S-M, and wherein each secondspecies is independently a molecule Y₁—([CH₂]_(t)—CH₂—O)_(n)—R₃—S-M,wherein M is the metal layer to which the species is bonded, and whereinX₁, Y₁, R₁, R₃, c, t, and n are independently as previously defined.